Autoimmune diseases such as multiple sclerosis (MS) may result from the failure of tolerance mechanisms to prevent expansion of pathogenic T cells. Our study is the first to establish that MS patients have abnormalities in FOXP3 message and protein expression levels in peripheral CD4+ CD25+ T cells (Tregs) that are quantitatively related to a reduction in functional suppression induced during suboptimal T-cell receptor (TCR) ligation. Of importance, this observation links a defect in functional peripheral immunoregulation to an established genetic marker that has been unequivocally shown to be involved in maintaining immune tolerance and preventing autoimmune diseases. Diminished FOXP3 levels thus indicate impaired immunoregulation by Tregs that may contribute to MS. Future studies will evaluate the effects of therapies known to influence Treg cell function and FOXP3 expression, including TCR peptide vaccination and supplemental estrogen.

CD4+CD25+ regulatory T cells (Treg cells) prevent T cell-mediated autoimmune diseases in rodents. To develop a functional Treg assay for human blood cells, we used FACS- or bead-sorted CD4+CD25+ T cells from healthy donors to inhibit anti-CD3/CD28 activation of CD4+CD25- indicator T cells. The data clearly demonstrated classical Treg suppression of CD4+CD25- indicator cells by both CD4+CD25(+high) and CD4+CD25(+low) T cells obtained by FACS or magnetic bead sorting. Suppressive activity was found in either CD45RO- (naive) or CD45RO+ (memory) subpopulations, was independent of the TCR signal strength, required cell-cell contact, and was reversible by interleukin-2 (IL-2). Of general interest is that a wider sampling of 27 healthy donors revealed an age- but not gender-dependent loss of suppressive activity in the CD4+CD25+ population. The presence or absence of suppressive activity in CD4+CD25+ T cells from a given donor could be demonstrated consistently over time, and lack of suppression was not due to method of sorting, strength of signal, or sensitivity of indicator cells. Phenotypic markers did not differ on CD4+CD25+ T cells tested ex vivo from suppressive vs. nonsuppressive donors, although, upon activation in vitro, suppressive CD4+CD25+ T cells had significantly higher expression of both CTLA-4 and GITR than CD4+CD25- T cells from the same donors. Moreover, antibody neutralization of CTLA-4, GITR, IL-10, or IL-17 completely reversed Treg-induced suppression. Our results are highly consistent with those reported for murine Treg cells and are the first to demonstrate that suppressive activity of human CD4+CD25+ T cells declines with age.

A T-cell receptor (TCR) peptide vaccine from the V beta 5.2 sequence expressed in multiple sclerosis (MS) plaques and on myelin basic protein (MBP)-specific T cells boosted peptide-reactive T cells in patients with progressive MS. Vaccine responders had a reduced MBP response and remained clinically stable without side effects during one year of therapy, whereas nonresponders had an increased MBP response and progressed clinically. Peptide-specific T helper 2 cells directly inhibited MBP-specific T helper 1 cells in vitro through the release of interleukin-10, implicating a bystander suppression mechanism that holds promise for treatment of MS and other autoimmune diseases.

Multiple sclerosis is an autoimmune disease in which T lymphocytes reactive to myelin basic protein (BP) could play a central role. T cells specific for BP were cloned from the blood of multiple sclerosis patients and normal individuals, and expression of T-cel receptor variable region genes was analyzed. A remarkable bias for use of f-chain variable region (VP) 5.2 and, to a lesser extent, Vj36.1 was seen among BP-specific clones from patients but not from controls. Multiple sclerosis (MS) is an immune-mediated disease characterized by central nervous system mononuclear cell infiltration and demyelination. Although the pathogenesis of MS is unknown, both genetic and environmental factors have been implicated in the disease process. Major elements ofthe genetic predisposition include an association of disease with particular class II major histocompatibility complex (MHC) haplotypes, in particular HLA-DR2 and -DQwl (1-5), as well as with certain polymorphisms within the T-cell receptor (TCR) a-chain and (3-chain gene complexes (6-9). These studies suggest that the disease involves CD4+ T cells bearing af3 TCR. In support of this idea, CD4+ T cells represent a major component of mononuclear cells in the brains of active patients (10), and limited sets of a-chain TCRs are present within central nervous system tissue of MS patients but not controls (11).T lymphocytes that recognize myelin basic protein (BP) have been shown to have potent demyelinating and encephalitogenic activity in animals (12-18). Accumulating evidence also suggests that BP-specific T cells may contribute to the pathogenesis ofMS. Thus, cells selected from MS patients on the basis of in vivo activation have specificity for BP (19). In addition, the frequencies of BP-reactive T cells are increased Monoclonal antibodies directed to these regions or synthetic peptides with sequences common to these TCR V regions can both protect and treat animals with clinical signs of experimental autoimmune encephalomyelitis (21,22,(25)(26)(27). For a similar approach to be applied to MS patients, it is critical to know if potentially pathogenic T cells also preferentially use a limited set of V region genes. In this manuscript, we analyze the TCR Va and Vf3 genes expressed in BP-specific T cells selected from the blood of MS patients and normal individuals. MATERIALS AND METHODSPatients. Blood samples were obtained from seven patients being followed at the Oregon Health Sciences University MS clinic. All patients had clinically and laboratory-supported definite MS with an average ambulation index of 3.2 + 1.7 (range 2-6) and an average Kurtzke disability status score of 3.8 + 2.0 (range 2-6). Four patients had relapsing/remitting disease, and three patients had chronic progressive disease. The normal individuals were from the Veterans Affairs Medical Center, selected on the basis of a positive proliferative response of peripheral blood mononuclear cells (PBMC) to human BP in culture (20). All subjects were HLA-typed by standard serological methods at the...

Prospects for specific immune intervention in T cell-mediated autoimmune disease via anti-idiotypic regulation depend on the degree of diversity of the responder cell antigen receptor repertoire. A highly heterogenous response against self epitopes offers little chance for such regulation. We report here that the Lewis rat autoimmune disease experimental allergic encephalomyelitis, generally considered to be a model of human multiple sclerosis, is caused by T cells that use a limited set of TCR V genes. We have cloned the rat TCR alpha and beta chain cDNAs from the Lewis rat x mouse T cell hybridoma 510, which retains the rat specificity for the encephalitogenic determinant of myelin basic protein (MBP). Using Northern blot analysis of T cell RNA with the cloned V region probes, we have found a specific, and near perfect, correlation between expression of TCR message hybridizing to the V alpha 510 and VB510 probes and specificity for the encephalitogenic determinant of MBP in both T cell hybridomas and encephalitogenic T cell clones. This restricted V gene usage provides a basis for observed idiotypic regulation of auto-reactive T cells, and possible therapy for autoimmune disease. A curious and unexplained observation is that the Lewis rat V alpha/V beta combination that dominates the encephalitogenic response to the 68-88 peptide of MBP is precisely the same V alpha/V beta combination used by the B10.PL mouse response to the encephalitogenic response to the 1-9 peptide of MBP.

Understanding the process of inducing T cell activation has been hampered by the complex interactions between APC and inflammatory Th1 cells. To dissociate Ag-specific signaling through the TCR from costimulatory signaling, rTCR ligands (RTL) containing the α1 and β1 domains of HLA-DR2b (DRA*0101:DRB1*1501) covalently linked with either the myelin basic protein peptide 85–99 (RTL303) or CABL-b3a2 (RTL311) peptides were constructed to provide a minimal ligand for peptide-specific TCRs. When incubated with peptide-specific Th1 cell clones in the absence of APC or costimulatory molecules, only the cognate RTL induced partial activation through the TCR. This partial activation included rapid TCR ζ-chain phosphorylation, calcium mobilization, and reduced extracellular signal-related kinase activity, as well as IL-10 production, but not proliferation or other obvious phenotypic changes. On restimulation with APC/peptide, the RTL-pretreated Th1 clones had reduced proliferation and secreted less IFN-γ; IL-10 production persisted. These findings reveal for the first time the rudimentary signaling pattern delivered by initial engagement of the external TCR interface, which is further supplemented by coactivation molecules. Activation with RTLs provides a novel strategy for generating autoantigen-specific bystander suppression useful for treatment of complex autoimmune diseases.

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Treatment with partial (p)MHC class II-β1α1 constructs (also referred to as recombinant T-cell receptor ligands – RTL) linked to antigenic peptides can induce T-cell tolerance, inhibit recruitment of inflammatory cells and reverse autoimmune diseases. Here we demonstrate a novel regulatory pathway that involves RTL binding to CD11b+ mononuclear cells through a receptor comprised of MHC class II invariant chain (CD74), cell-surface histones and MHC class II itself for treatment of experimental autoimmune encephalomyelitis (EAE). Binding of RTL constructs with CD74 involved a previously unrecognized MHC class II-α1/CD74 interaction that inhibited CD74 expression, blocked activity of its ligand, macrophage migration inhibitory factor, and reduced EAE severity. These findings implicate binding of RTL constructs to CD74 as a key step in both antigen-driven and bystander T-cell tolerance important in treatment of inflammatory diseases.